Expression, Purification, and Initial Characterization of the Recombinant Storage Protein Precursor ofTheobroma cacao |
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Authors: | Juli O'Connor Dean F Revell Karen E Masters Ian F Connerton Ian G Sumner |
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Institution: | aDepartment of Protein Engineering, BBSRC Institute of Food Research, Reading Laboratory, Earley Gate, Whiteknights Road, Reading, RG6 2EF, United Kingdom;bRSSL, The Lord Zuckerman Research Centre, The University, Whiteknights, P.O. Box 234, Reading, RG6 2LA, United Kingdom |
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Abstract: | The gene encoding the 67-kDa cocoa storage protein precursor has been cloned fromTheobroma cacaoand expressed inEscherichia coliusing the pET expression system. The recombinant storage protein has been renatured from inclusion bodies at 30°C using 20 m
glycine–NaOH buffer, pH 10.0, containing 1 m
oxidized glutathione and 0.1% Brij. The renatured protein was purified and demonstrated to adopt a stable native conformation by optical spectroscopy. Secondary structure analysis from circular dichroism indicated the protein to be 23% α-helix and 38% β-sheet, in close agreement with values obtained using a secondary structure prediction program. |
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